Panoply™ Human NFKB1 Over-expressing Stable Cell Line

Targeted protein degradation has emerged as a potent therapeutic strategy for degrading disease-associated targets. While the design of proteolysis-targeting chimeras (PROTACs) is highly modular, the discovery of "molecular glue" degraders presents a greater challenge. Here, researchers combined phenotypic screening of a covalent ligand library with chemoproteomic approaches to rapidly identify a covalent molecular glue degrader and elucidate its mechanism of action. They identified EN450, a cysteine-reactive covalent ligand that inhibits leukemia cell viability in a manner dependent on NEDDylation and the proteasome. Chemoproteomic analysis revealed that EN450 covalently interacts with C111, an allosteric site on the E2 ubiquitin-conjugating enzyme UBE2D, while quantitative proteomics identified the oncogenic transcription factor NFKB1 as a potential degradation target. In summary, this study identifies a covalent molecular glue degrader that triggers the degradation of a transcription factor in cancer cells by uniquely inducing a proximity-based interaction between the E2 enzyme and the transcription factor.

Chemical proteomic analysis identified approximately 80 potential cysteine ​​targets, indicating that EN450 is clearly not a selective compound. Researchers sought to link the degradation of NFKB1-a known oncogenic transcription factor-to the mechanism by which EN450 inhibits cell proliferation. In HAP1 cells stably overexpressing NFKB1, the anti-proliferative effect mediated by EN450 was significantly attenuated in a dose-dependent manner (Figure 1a-b). Researchers also transiently transfected NFKB1 into HEK293T cells; the results showed that, following EN450 treatment, both the impairment of cell viability and the reduction in NFKB1 levels were dependent on NEDDylation (Figure 1d-e). Furthermore, NFKB1 overexpression exerted a more pronounced rescue effect against EN450-mediated anti-proliferative activity (Figure 1f). Although other polypharmacological mechanisms may contribute to the anti-proliferative action of EN450, existing data suggest that NFKB1 is at least partially involved in the compound's anti-cancer effects.

Figure 1. Understanding the role of NFKB1 in EN450-mediated effects. (King E A, et al., 2023)